The emergence of unnatural DNA bases provides opportunities to demystify the mechanisms how DNA polymerases faithfully decode chemical information on the template. For instance, we previously showed that two unnatural cytosine bases (termed "M-fC" an ...

The emergence of unnatural DNA bases provides opportunities to demystify the mechanisms how DNA polymerases faithfully decode chemical information on the template. For instance, we previously showed that two unnatural cytosine bases (termed "M-fC" and "I-fC"), which are chemical labeling adducts of the epigenetic base 5-formylcytosine, can induce C-to-T transition during DNA amplification. However, how DNA polymerases recognize such unnatural cytosine bases remain enigmatic. Herein, we determined crystal structures of the unnatural cytosine bases pairing to dA/dG in the KlenTaq-host-guest complex system and pairing to dATP in the active site. The results show that both M-fC and I-fC base pair with dA/dATP, but not with dG, in a Watson-Crick geometry. Our study reveals that the formation of the Watson-Crick geometry, which may be enabled by the A-rule, is important for the recognition of unnatural cytosines, and this expands the current understanding of unnatural base recognition by DNA polymerases.